Preparation of an anti-hog cholera product



PREPARATION OFAN ANTI-HOG -CHOLERA :rnonucr Mark E. Davenport,-.'Jr.,LaGrange, Irby Bunding, Chicago, and'Maurice A. Schooley, WesternSprings, 111., assignors to Armour and Company, Chicago, 111., acorporation of Illinois No Drawing. Application July 29, 1957 Serial No.674,593

4 Claims. (Cl. 167-80) This application is a continuation-in-part.ofpending application, Serial No. 539,002,.filed October 6, 1955, andnow abandoned. I

This invention relatesto .the preparation of an antihog cholera product,and more particularly .to a process for obtaining a concentrate of hogcholera antibodies suitable for producing in swine a positive immunityto hog cholera virus.

The anti-hog cholera produotutilized in conventional immunization ofswine is anti-hog cholera serum pre-.

pared according to the process specified in the rules and regulations ofthe U.S. Department of Agriculture. This process involves injecting intohogswhich have acquired an immunity to hog cholera at Ieast S cc. ofvirulent hog cholera-virus per pound of hog body weight. The immunityto'hog cholera 'can be acquired by the animal as a natural immunity orproduced. by v simultaneous injection :of virulent or modified virus andanti-hog cholera serum into the animal. Hog cholera immune hogs-injected'with'virulentonmodified hog cholera virus develop hyperimmunization,i'.e., they produce an excess of hog cholera antibodies. After a periodof about ten to fourteen days of incubation the hyperimmunized blood"canbe withdrawn from'theanimal. "Several bleedings can be obtained overa period "of not more than forty days after'hyperimmunizationdn whicheachbleeding is at least seven days from the preceding one. The usualsequenceof bleedings includes three tail bleedings, and after-finallysacrificing the hog, a throat-bleeding. The hyperimmunized blood canthen--be pooled and, after defibrination, the serum canbeseparatedfromthe stroma and-pasteurized; "The resulting anti-hog cholera serum can bepreserved with phenol andtested'for potency by injection into non-immunehogs together with virulent hog cholera virus.

Although it 'is recognized ,that a large part of the cost of -a.commercial anti=hog cholera serum is due to the large hyperingfdoses'of5 cc. of virulenthog cholera virus which is initially injected for eachpound of the immune hog body weight, nevertheless the industry has heldto the belief for the .past half-century that at least 5 cc. of thevirus:per. pound of body weight is essential, and the regulations of theBureau :of Animal Industry have been based on the 5 cc. dose throughoutthe entire period until the present time.

An object of this invention is to provide a process for concentratinghog cholera antibodies to obtain an antihog cholera product which canconfer immunity upon hogs by theinjectionof a significantly smallerquantity of such product than washeretofore possible. Another object isto provide a process for preparing an anti-hogcholera,:productadaptable.to .large scale manufacturing withconsiderably reduced costs in: comparison with prevailing practices. :.Afurther 'object is to providean antihog cholera product of-.a 1potencyin-the order of three or four times as great as thatof-antrhog choleraserum.

nited States Patent 0 ice Otherobjects and advantages will becomeapparent as the specification proceeds.

In one aspect of this invention the anti-hog; cholera product can beobtained by exposing -.a hog cholera immune hog to virulent hog choleravirus in'an amount equivalent to not more than 4.5 cc. of hog cholera-,infected'blood per pound of hog body weight to hyper- .immunize thehog. The blood of the'hyperimmunized hog .can .be thenwithdrawn andsubjected ,tochet iical fractionation to obtain a concentratenof.thehogcholera antibodies therein suflicient to produce immunity,ingnonimmune hogs. This chemical fractionation ..can'be,obtained byextracting from the hyperimmunized blood the serum portion thereof, andtreating suchserum with a protein-precipitating reagent to.selectivelyremovea fraction thereof consisting principally ofserumglobulins and hog cholera antibodies.

Although the hog choleravirus preparation utilizedin this process toproduce hyperimmunization .ofan immune .hog may be modified orattenuated, v we have obtained lespeciallydesirable results withvirulent hog cholera virus.

By hog cholera-infected blood -is meant virulenthog cholera virus orhyperimmunizing virus obtained according to the process specified in theRules andRegulations .Relating to Viruses, Serums, etc., promulgated bythe US. Department of Agriculture,-March l, 1949, and. specified insection 118 thereof. By modified or .attenuated hog cholera virus ismeant a hog cholera virus which has been altered from adiseaserproducing virus to one incapable of causing-disease. lorexample, a modified or attenuated hogcholeravirus' can be prepared bythe-method described in the .co-pending =patentqapplication of J. A.Baker, Serial No. 486,676, filed-February 7, 1955, wherein the hogcholera virus is rabbit-modified and hog-propagated and wherein apreferred practice of the attenuation process involves inoculating anonimmune pig from 5 to 10 weeks in age with amattenuated hogcholeravirus, feeding the inoculated-gpig an adequate .cholera virusfrom the pig when its temperature has droppedfrom a maximum level abovenormal to an intermediate level above normal, providing the pig 'hasapproached a weight gain above normal of about 4--to 5 pounds per week.

The hyperimmunization of a hog cholera. immune hog .in this processshouldbe produced by the inoculation thereof with the hog cholera virusin an amount equiv- -;-alent; to .not more than 4.5 cc.. of hogcholera-infected .zbloodper pound of hog body weight, i.e., theequivalent .of from 0.002 to 4.5 cc. of hog cholera-infected bloodper-pound of hog body weight. We have found that ibetter results areobtained when the hog cholera immune hog is inoculated with the hogcholera virus in an amount equivalent to 0.02 to 3.0 cc. of hogcholera-infected blood per pound of hog body weight, and an especiallydesirable hog. cholera product can be produced when hyperimmunization isobtained by the inoculation'of a .hog cholera immune hog with the hogcholera virus in an amount equivalent to from 0.1 to 1.0 cc. ,of hogcholera-infected blood per pound of hog body weight.

The inoculation of the hog cholera immune hog with hog cholera virus canbe obtained by parenteral administration thereof, e.g., intravenous orsubcutaneous injection.

The period of incubation for obtaining hyperimmunization of the hog canbe at least 11 days after the hyperimmunizing inoculation of hog choleravirus. Also, the number and sequence of the blood withdrawals from thehyperimmunized hog can be in accordance with'prevailing practices. Forexample, the Withdrawal of bloodfrom the hyperimmunized choleraantibodies.

hog can be obtained in from one to four bleedings at various timeintervals from the tail, vein, throat, etc. This recovered blood canthen be chemically fractionated to extract a concentrate of 'hog choleraantibodies and leaving behind the fibrinogen,

albumin and other contaminant substances. This extraction of the hogcholera antibody concentrate may be obtained by defibrinating the blood,separating the stroma from the defibrinated blood to produce serum,

and then fractionating the serum with a protein-precipitating agent toobtain a fraction thereof consisting principally of the serum globulinsand'hog cholera antibodies.

'The defibrination of the blood can be produced by stir- 'ring the bloodwith a wire to provide conversion of fibrinogen to fibrin, and thenseparating the coagulated fibrin from the blood. The separation ofhemoglobin and cellular debris from the defibrinated blood can beachieved by adding thereto a Rouleauxing agent, such as I jack beanextract, to obtain agglutination of the red blood cells, mixing alsowith the defibrinated blood a saturated solution of sodium chloride inan amount equivalent to 3 cc. per 100 cc. of blood, and centrifuging orfiltering the "blo'od to separate the resulting precipitate.

' by R. J. Seidel and M. J. West, U.S. Patent application Serial No.406,598, filed January 27, 1954, now Patent No. 2,785,105, wherein onemethod of preparing a hog cholera antibody concentrate involvesselectively coagulating a globulin fraction of serum containing gammaglobulins, beta globulins and hog cholera antibodies by adjustinghyperimmunized serum to a pH of from 4.8 to 6.0, heating such serum at atemperature within the range of 40 to 65 C. to obtain a coagulum of suchglobulins and separating the coagulum from the solution of hog Othersuitable methods for chemical fractionation of the hyperimmunized seruminvolve precipitation with ammonium sulfate, heavy metal ions,

- etc.

In the preferred method of obtaining the anti-hog cholera producthyperimmunized serum can be adjusted to a pH of from 6.7 to 7.1 and toan alcohol concentration of from 18 to 30% by volume, and the resultingprecipitate may be separated from the supernatant liquid. The ionicstrength of this alcohol solution'may be from about 0.1 to 0.2 and theadjustment of such ionic strength can be obtained by regulating theamount of sodium chloride added to the defibrinated blood in separatingout the cellular material. The temperature during this fractionationstep should be from about 5 C. we temperature above the freezing pointof the alcohol solution. The alcohol, utilized in this fractionationprocedure, can be methanol or ethanol, although ethanol is to bepreferred, and can be added to the hyperimmunized serum in substantialanhydrous form, e.g. denatured 95% ethanol, or in a more dilute state toprevent denaturation of the protein therein, e.g. 53% aqueous alcohol.Especiallly desirable results are achieved when the alcoholfractionation is produced by adjusting the hyperimmunized serum to a pHof about 6.9, an ethanol concentration of about 25% by volume, and ionicstrength of about 0.15, and a temperature of about to C. The precipitatethereby produced contains, principally, serum globulins and hog choleraantibodies. This precipitate can be separated from the supernatantliquid and suspended in water and the resulting solution adjusted to apH of about 7.2 to precipitate other contaminant proteins, principallybeta globulins. This beta globulin precipitate can be separated bycentrifugation. The soluble fraction, containing principally gammaglobulins and hog cholera anti- 4 bodies, can be mixed with apreservative, e.g. phenol in an amount equivalent to 0.5%, and packagedfor use in producing passive immunity in non-immune hogs.

The defibrinated serum, prior to chemical fractionation, can bepasteurized by heating to a temperature of from 58 to 59 C. in asuitable pasteurizing apparatus. When pasteurization has been completedthe serum should be cooled as quickly as possible, and then mixed with apreservative, such as phenol in an amount equivalent to 0.5% and storedpending the chemical fractionation operation. Also, the anti-hog choleraproduct obtained in this method may be similarly pasteurized andpreserved.

The potency or titer of this anti-hog cholera product can be ascertainedby inoculating non-immune hogs with such product in combination withvirulent hog cholera virus, and observing thedegree'of protectionafforded the hogs against hog cholera in comparison with control hogswhich are inoculated with virulent hog cholera virus in the absence ofhog cholera antibody concentrate.

This anti-hog cholera product can be utilized in producing immunityagainst hog cholera by inoculating nonimmune hogs concomitantly withvirulent or modified hog cholera virus and such product.

This invention can be further illustrated by the following specificexamples:

EXAMPLE I The following data will demonstrate the advantage of smalldoses of hyperimmunizing virus combined with fractionation of theresultant serum over the existing method of preparing serum.

Twelve hog cholera immune hogs were obtained from each of three sources.Six hogs were randomly selected from each group and hyperimmunized witha standard dose of virus, 5 cc. per pound of body weight. The remainingsix hogs in each group were hyperimmunized with a low dose of virus, 0.5cc. per pound of body weight.

The control groups were handled at all times in a standard fashion asprescribed by the US. Department of Agriculture. They were tail bled onthe 11th, 18th and 25th days following hyperimmunization, and sacrificedfor complete bleeding on the 32nd day. The serum obtained was processedunder standard conditions.

The testgroups were tail bled on the 11th, and 18th days followinghyperimmunization and sacrificed for complete bleeding on the 25th day.The serum obtained was subjected to chemical fractionation.

The fiinal yields of potent sera are shown in Table 1. Groups A-l, C-1and E-1 were control groups, and

A2, C-2 and B-2 were the respective test groups.

otal Yield of Yield of Total Amt. Yield Standard Standard of HyperlmofStd. Potency Potency Group No. Weight munizlng Potency Serum Serum/cc.of

Virus (cc.) erum per 1b. ype (0a.) (00.) munlztng Virus (cc) A-l 1,5,775 31, 306 27.0 5.4 O-l 1, 145 5, 725 32, 795 28. 6 5. 7 E-l l, 3856,925 34,616 25.0 5.0

Total"..- 3, 685 18,425 98,717 26. 8 5. 4

A-2 1,255 627. 5 21,992 17.5 35.0 C-2 l, 585. 0 252 18. 2 36. 3 E-2 1,425 712. 5 22, 294 15. 6 31. 3

TotaL 3, 850 1, 925 0 65, 538 17. 5 34. 0

All six sera were shown by assay in hog cholera susceptible hogs to beof equal potency.

A 90% decrease in the dose of hyperimmunizing virus combined withfractionation of the serum resulted in a decrease of only 41.3% in serumyield. The yield of potent serum per cc. of hyperimmunizing virus wasincreased from 5.4 cc. to 34.0 cc.

EXAMPLE II Thet following method was utilized in preparing an anti-hogcholera product:

.Hog cholera immune hogs were randomly selected .from three .sourceshaving .an average weight, respectively, .of 175, 280 and 2-60 .pounds.There were five hogs .from each source :resulting .in a hog choleraimmune-hog .group numbering 15. These hogs were inoculated with 1J0 cc.of virulent hog cholera virus per pound of hog body weight, to producehyperimmunization .thereof. The hogs were bled from the tail on thetwelfth .day after inoculation with virus and bled from the throat onthe nineteenth day after inoculation. The blood obtained in eachbleeding was immediately defibrinated and 0.5% of phenol added thereto.The defibrinated blood from each bleeding was pooled and combined withjack bean extract and 3 cc. of a saturated sodiumchloride'solution per100 cc. of blood. The resulting mixture was centrifuged to separate thestroma from the serum. This serum was pasteurized by heating at atemperatureof 58 to 590 C. for a period of thirty minutes, then quicklycooled, mixed with phenol "in an amount equivalent to 0.5%, andmaintained in storage for subsequent chemical fractionation.

EXAMPLE III The hyperimmune serum obtained according to the method ofExample II was adjusted to pH 6.9, and a 53% aqueous :solution of 3Aalcohol added thereto, capillarywise, to a final alcohol concentrationof 25% by volume. The alcohol was chilled to a temperature of about 20C. prior to addition to the serum, and the resulting alcohol solutionmaintained at a tempera ture of about 5 C. The resulting precipitate wasseparated from the supernatant liquid by centrifugation at a temperatureof 5 C. and held at refrigerator temperature pending further treatment.

EXAMPLE IV A portion of the precipitate obtained according to the methodof Example III was suspended in water, in an amount such as to produce afinal protein concentration of about 3%. The resulting suspension wasadjusted to pH 7.2 and stirred for a period of 24 hours at a temperatureof about 0 C. The precipitate thereupon formed was separated from thesupernatant liquid by centrifugation. To this supernatant liquid wasadded phenol in an amount equivalent to 0.5% and the resulting anti-hogcholera product stored pending inoculation into non-immune hogs.

EXAMPLE V A portion of the precipitate obtained according to the methodof Example III was suspended in water in such amount as to produce afinal protein concentration of about 6%. This solution was adjusted topH 7.2 and stirred for a period of 24 hours at a temperature of about 0C. The precipitate thereupon formed was separated from the supernatantliquid by centrifugation. To this supernatant liquid was added phenol inan amount equivalent to 0.5%, and the resulting anti-hog cholera productstored pending inoculation into non-immune hogs.

EXAMPLE VI A portion of each of the anti-hog cholera products obtainedby the methods of Examples IV and V were pasteurized by heating at atemperature of 58 to 59 C. for a period of 30 minutes, then quicklycooled and phenol in the amount of 0.5 added thereto for preservativepurposes. The pasteurized product of Example IV was designated as IV-A,while the pasteurized prod net of Example V was designated as V-A.

EXAMPLE VII The potency of the anti-hog cholera products obtained by theprocesses of the foregoing examples was compared with that of commercialanti-hog cholera serum (VH). These anti-hog cholera products aredesignated herein by the number of the example according-to which theywere produced.

Non-immune pigs, numbering 123, were divided into 8 groups, each groupconsisting of 15 or 16 pigs. Each pig was inoculated with 2 cc. 'ofvirulent hog cholera virus, and each groupof pigs, with the exceptionofone group which was maintained as a control, was injected with one ofthese anti-hog cholera products. Within each group of pigs theparticular anti-hog cholera product was injected at two dosage levels.The mean temperature of the pigs on the day-of treatment was 102.30 1515andF the standard deviation of temperatures was The pigs'were' observedfor a period of l tdays following inoculation, and the reactions of eachpig within the groups recorded according to the following scoringsystem: I

Definition Not evgn anelevated temperature during the 14-day test perioA temperature of 104.0 Faor higher on at least one day during the 14dayperiod; no other symptoms. visibly slow on at least one day during thetest period but but not sick; wellatthe-end of 14 days. Vis1bly sick onat least one day during the test period; well at the end of 14 days.Slow or sick at the end of l4 days; well later.

'Slow or sickat the end of 14 daysymorlbund or dead later. Moribund atthe .end of 14 days .(killed). "Dead on day 12, 13 or I4. Dead on day 9,10 or 11. Dead on day 6, 7 or 8.

The results are recorded in the following table:

Score Anti-Hog Cholera Product Dose Mean (ca) Score Control (virus only)1 K indicates that the pig died.

A mean score in this table of not more than 3.0 indicates a negativeresponse, while a score of greater than 3.0 indicates a positiveresponse. Therefore, these results demonstrate that anti-hog choleraproducts obtained by the process of this invention have an activity atleast as high, and a potency in the order of twice as great, as that ofanti-hog cholera serum.

EXAMPLE VIII The chemical fractionation procedure set forth in ExampleIII was utilized in the treatment of hyperimmune serum obtained byinoculating hog cholera immune hogs with 0.002 cc. and 0.02 cc. ofvirulent cholera virus per pound of hog body weight. The resultingantihog cholera products were evaluated in non-immune hogs according tothe method of Example VII. The results indicated that the hog choleraantibody potency of these products was approximately twice as great asthat of commercial anti-hog cholera serum (VII).

While in the foregoing specification various embodiments of thisinvention have been set forth and specific detailsthereof elaborated forthe purpose of illustration, it will be apparent to those skilled in theart that the invention issusceptible to other embodiments and that manyof these details can be varied widely without departing from the basicconcept and spirit of the invention.

We claim:

1. In a process for preparing an anti-hog cholera product, the step ofproducing hyperimmune blood, from which said anti-hog cholera productcan be made, by exposing a hog cholera immune hog to hog cholera virusin a total amount equivalent from about 0.02 to 3.0 cc. of hogcholera-infected blood per pound of hog body weight to hyperimmunizesaid hog, and subsequently withdrawing blood from said hyperimmunizedhog.

2. In a process for preparing an anti-hog cholera product, the step ofproducing hyperimmune blood, from which said anti-hog cholera productcan be made, by exposing a hog cholera immune hog to hog cholera virusin a total amount equivalent from about 0.1 to 1.0 cc. of hogcholera-infected blood per pound of hog body weight to hyperimmunizesaid hog, and subsequently withdrawing blood from said hyperimmunizedhog.

3. In a process for preparing an anti-hog cholera product, byinnoculating a hog cholera immune hog with virulent hog cholera virus tohyperimmunize said hog, withdrawing blood from said hyperimmunized hog,extracting serum from said blood, concentrating the antibodies in saidserum by chemically precipitating an antibody concentrate, andrecovering said concentrate, the improved step comprising exposing saidhog cholera immune hog to said hog choleravirus in a total amountequivalent from about 0.02 to 3.0 cc. of hog cholerainfected blood perpound of hog body weight to hyperimmunize said hog.

5 g 4. In a process for preparing an anti-hog cholera product, byinoculating a hog cholera immune hog with virulent hog cholera virus tohyperimmunize said hog, withdrawing blood from said hyperimmunized hog,extracting serum from said blood, adjusting said serum to 10 a pHof.from 6.7 to 7.1 and to an alcohol concentration of from about 18 toabout 30% by volume and separating the precipitate thereupon formed fromthe supernatant liquid, the improved step comprising exposing said hogcholera immune hog tosaid hog cholera 15 virus in a total amountequivalent from about 0.1 to 1.0 cc. of hog cholera-infected blood perpound of.hog body weight to hyperimmunize said hog.

References Cited in the file of this patent OTHER REFERENCES King: Art.in Tech. Bull., Kansas State Agr. Coll., Exp. Stat., Bull. 171, pp.168184 (mp. p. 170), September 1910.

30 F.B., Farmers Bulletin 834, US. Dept. of Agri., January 1921, p. 16.i

1. IN A PROCESS FOR PREPARING AN ANTI-HOG CHOLERA PRODUCT, THE STEP OFPRODUCING HYPERIMMUNE BLOOD, FROM WHICH SAID ANTI-HOG CHOLERA PRODUCTCAN BE MADE, BY EXPOSING A HOG-CHOLERA IMMUNE HOG TO HOG CHOLERA VIRUSIN A TOTAL AMOUNT EQUIVALENT FROM ABOUT 0.02 TO 3.0 CC. OF HOGCHOLERA-INFECTED BLOOD PER POUND OF HOG BODY WEIGHT TO HYPERIMMUNIZESAID HOG, AND SUBSEQUENTLY WITHDRAWING BLOOD FROM SAID HYPERIMMUNIZEDHOG.